Hypoxic areas of poorly vascularized tumors presumably limit the effectiveness of cancer therapy, yet convenient methods to measure either the number of hypoxic cells or the degree of cellular oxygen deprivation are not available. We have recently found that flow cytometry techniques may be applicable to this problem. Our work has used fluorescent nitroheterocycles, which are preferentially metabolized and bound in hypoxic cells, or non-toxic fluorescent stains which penetrate slowly into spheroids so that intracellular stain intensity decreases with depth (as does oxygenation). We expect to further develop these approaches in conjunction with fluorescence-activated cell sorting, to quantitate the oxygenation of individual cells of tumors. We first plan to select improved hypoxic cell probes by examining cellular uptake and binding of several fluorescent nitroheterocycles under defined oxygen tensions in vitro. Our ability to identify hypoxic cells using these agents will be evaluated by independently exposing thioguanine-sensitive and resistant V79 cells at different oxygen concentrations, then mixing the cells, asceptically sorting on the basis of fluorescence, and measuring cell growth in standard and selective medium. Subsequently, promising nitroheterocycles and slowly-penetrating fluorescent stains will be evaluated in spheroids, and then in transplantable tumors in mice. In these systems, survival of stained, sorted cells following irradiation will be used to determine the sensitivity and accuracy of our hypoxic cell identification procedures. We believe that our techniques will quantify the number of hypoxic tumor cells as well as indicate the degree of oxygenation of individual cells, and will thus provide a basis for the eventual development of a non-invasive methodology (eg, radio-labelled agents) suitable for use in human tumors.